FokI-RYdCas9 Mediates Nearly PAM-Less and High-Precise Gene Editing in Human Cells.

The demand for high-precision CRISPR/Cas9 systems in biomedicine is experiencing a notable upsurge. The editing system fdCas9 employs a dual-sgRNA strategy to enhance editing accuracy. However, the application of fdCas9 is constrained by the stringent requirement for two protospacer adjacent motifs (PAMs) of Cas9. Here, we devised an optimized editor, fRYdCas9, by merging FokI with the nearly PAM-less RYdCas9 variant, and two fRYdCas9 systems formed a dimer in a proper spacer length to accomplish DNA cleavage. In comparison to fdCas9, fRYdCas9 demonstrates a substantial increase in the number of editable genomic sites, approximately 330-fold, while maintaining a comparable level of editing efficiency. Through meticulous experimental validation, we determined that the optimal spacer length between two FokI guided by RYdCas9 is 16 base pairs. Moreover, fRYdCas9 exhibits a near PAM-less feature, along with no on-target motif preference via the library screening. Meanwhile, fRYdCas9 effectively addresses the potential risks of off-targets, as analyzed through whole genome sequencing (WGS). Mouse embryonic editing shows fRYdCas9 has robust editing capabilities. This study introduces a potentially beneficial alternative for accurate gene editing in therapeutic applications and fundamental research.

Unveiling the hidden role of the interaction between CD36 and FcγRIIb: implications for autoimmune disorders.

BACKGROUND: The role of the scavenger receptor CD36 in cell metabolism and the immune response has been investigated mainly in macrophages, dendritic cells, and T cells. However, its involvement in B cells has not been comprehensively examined. METHODS: To investigate the function of CD36 in B cells, we exposed Cd36fl/flMB1cre mice, which lack CD36 specifically in B cells, to apoptotic cells to trigger an autoimmune response. To validate the proteins that interact with CD36 in primary B cells, we conducted mass spectrometry analysis following anti-CD36 immunoprecipitation. Immunofluorescence and co-immunoprecipitation were used to confirm the protein interactions. RESULTS: The data revealed that mice lacking CD36 in B cells exhibited a reduction in germinal center B cells and anti-DNA antibodies in vivo. Mass spectrometry analysis identified 30 potential candidates that potentially interact with CD36. Furthermore, the interaction between CD36 and the inhibitory Fc receptor FcγRIIb was first discovered by mass spectrometry and confirmed through immunofluorescence and co-immunoprecipitation techniques. Finally, deletion of FcγRIIb in mice led to decreased expression of CD36 in marginal zone B cells, germinal center B cells, and plasma cells. CONCLUSIONS: Our data indicate that CD36 in B cells is a critical regulator of autoimmunity. The interaction of CD36-FcγRIIb has the potential to serve as a therapeutic target for the treatment of autoimmune disorders.

GPR43 regulates marginal zone B-cell responses to foreign and endogenous antigens.

Marginal zone (MZ) B cells are innate-like B cells that produce polyreactive antibodies with an affinity for microbial molecular patterns and carbohydrate ligands. MZ B cells have been shown to be important in mediating immunity to various bacteria including Streptococcus pneumoniae and are also implicated in inflammatory syndromes including lupus erythematosus. The intestinal microbiota is responsible for producing short-chain fatty acids, which can regulate immune cell function by several mechanisms including ligation of the G-protein-coupled receptor (GPR)43. Herein, we show that MZ B cells express Gpr43 messenger RNA and that the absence of this receptor impacts on MZ B-cell surface marker expression and antibody production. In T-cell-independent responses to the hapten 4-hydroxy-3-nitrophenylacetic acid (NP), mice deficient in GPR43 displayed higher serum titers of NP-specific antibodies. Moreover, in response to a pneumococcal polysaccharide vaccine, GPR43-deficient mice developed robust serum antibody responses and had markedly increased numbers of splenic antibody-secreting cells, compared with control mice. Finally, serum immunoglobulin M autoantibodies to double-stranded DNA and phosphatidylcholine were increased in resting 10-15-week-old mice lacking GPR43. Taken together, mice lacking GPR43 have heightened antibody responses to T-cell-independent antigens, which may be a result of impaired regulation of MZ B cells.

The B cell response to both protein and nucleic acid antigens displayed on apoptotic cells are dependent on endosomal pattern recognition receptors.

In systemic autoimmune diseases such as systemic lupus erythematosus (SLE), B cell tolerance is lost and there is a production of autoantibodies that drive pathology. The specificities of these antibodies are towards a wide range of autoantigens including proteins such as serum factors including cytokines as well as towards nucleic acids and modified glycolipids. It is known that endosomal pattern recognition receptors are involved in specific responses but if they drive specificity towards a specific group of autoantigens is not known. Here, we used syngeneic apoptotic cells alone to break B cell tolerance and investigated the antibody response in Unc93b1 mutant mice that lack signalling from the TLR3, TLR7 and TLR9 receptors. We found that specific B cell responses known from patients with SLE including antibodies towards Ro-52/60, La, cardiolipin as well as DNA were all significantly lower in the knockout mice. Thus, we found that endosomal TLR receptors were involved in break of tolerance and drive B cell responses for protein, nucleic acid and modified lipid antigens. This pinpoints these receptors as key drivers for the full range of antibody driven pathology in SLE and suggests that targeting of endosomal TLR driven responses will quench all B cell driven autoreactivity.

Genotype distribution of Chinese Mycoplasma bovis isolates and their evolutionary relationship to strains from other countries.

This study was undertaken to determine the genotypic distribution of Chinese M. bovis strains and their similarity to isolates from other countries. Two multilocus sequence typing (MLST) schemes (MLST-1 and MLST-2) and pulsed field gel electrophoresis (PFGE) were used to compare 44 Chinese strains and the M. bovis type strain PG45. The results showed a high genetic homogeneity of Chinese isolates; 43 of 44 (97.7%) Chinese isolates were identified as ST-10 and as ST-34 by MLST-1, while for MLST-2 42 of 44 (95.5%) were identified as ST-10 with the two remaining isolates of ST-32 and ST43. PFGE clustered 42 of 44 (95.5%) of the Chinese isolates into PT-I. The overall agreement rate between the three typing methods was 97.8% (95% CI:86.8-99.9%). The type strain PG45 was identified as a unique type by all three methods. When the MLST-2 scheme was further used to analyze 16 isolates of Australian and Israeli origin ST-10 was more dominant among Australian isolates (7/8), compared with those from Israel (3/8). The evolutionary relationship of the 60 isolates typed in this study assessed together with 206 additional isolates retrieved from pubmlst/mbovis database analyzed by geoBURST Minimum spanning tree (MST) confirmed that the Chinese, Israeli and Australian M. bovis isolates typed in this study that were predominantly ST-10, were clustered in CC3 with isolates originating from the USA. Our results suggest that ST-10 is an emerging clone of M. bovis population. We hypothesized that the widespread distribution of this type is a result of global livestock movements. These findings will help further the understanding of the global evolution of M. bovis and development of novel vaccines against M. bovis.

Indirubin Treatment of Lipopolysaccharide-Induced Mastitis in a Mouse Model and Activity in Mouse Mammary Epithelial Cells.

Indirubin is a Chinese medicine extracted from indigo and known to be effective for treating chronic myelogenous leukemia, neoplasia, and inflammatory disease. This study evaluated the in vivo anti-inflammatory activity of indirubin in a lipopolysaccharide- (LPS-) induced mouse mastitis model. The indirubin mechanism and targets were evaluated in vitro in mouse mammary epithelial cells. In the mouse model, indirubin significantly attenuated the severity of inflammatory lesions, edema, inflammatory hyperemia, milk stasis and local tissue necrosis, and neutrophil infiltration. Indirubin significantly decreased myeloperoxidase activity and downregulated the production of tumor necrosis factor-α, interleukin-1ß (IL-1ß), and IL-6 caused by LPS. In vitro, indirubin inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner. It also downregulated LPS-induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein and inhibitor of kappa B. In addition to its effect on the NF-κB signaling pathway, indirubin suppressed the mitogen-activated protein kinase (MAPK) signaling by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), P38, and c-jun NH2-terminal kinase (JNK). Indirubin improved LPS-induced mouse mastitis by suppressing TLR4 and downstream NF-κB and MAPK pathway inflammatory signals and might be a potential treatment of mastitis and other inflammatory diseases.

Development of a universal antibiotic resistance screening system for efficient enrichment of C-to-G and A-to-G base editing.

To expand the scope of genomic editing, a C-to-G transversion-based editor called CGBE has been developed for precise single-nucleotide genomic editing. However, limited editing efficiency and product purity have hindered the development and application of CGBE. In this study, we introduced the Puromycin-Resistance Screening System, referred to as CGBE/ABE-PRSS, to select genetically modified cells via the CGBE or ABE editors. The CGBE/ABE-PRSS system significantly improves the enrichment efficiency of CGBE- or ABE-modified cells, showing enhancements of up to 59.6 % compared with the controls. Our findings indicate that the CGBE/ABE-PRSS, when driven by the CMV promoter, results in a higher enrichment of edited cells compared to the CAG and EF1α promoters. Furthermore, we demonstrate that this system is compatible with different versions of both CGBE and ABE, enabling various cell species and simultaneous multiplexed genome editing without any detectable random off-targets. In conclusion, our developed CGBE/ABE-PRSS system facilitates the selection of edited cells and holds promise in both basic engineering and gene therapy applications.

Structure-guided discovery of highly efficient cytidine deaminases with sequence-context independence.

The applicability of cytosine base editors is hindered by their dependence on sequence context and by off-target effects. Here, by using AlphaFold2 to predict the three-dimensional structure of 1,483 cytidine deaminases and by experimentally characterizing representative deaminases (selected from each structural cluster after categorizing them via partitional clustering), we report the discovery of a few deaminases with high editing efficiencies, diverse editing windows and increased ratios of on-target to off-target effects. Specifically, several deaminases induced C-to-T conversions with comparable efficiency at AC/TC/CC/GC sites, the deaminases could introduce stop codons in single-copy and multi-copy genes in mammalian cells without double-strand breaks, and some residue conversions at predicted DNA-interacting sites reduced off-target effects. Structure-based generative machine learning could be further leveraged to expand the applicability of base editors in gene therapies.

In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function.

ABSTRACT: Bruton's tyrosine kinase (BTK) is an enzyme needed for B-cell survival, and its inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has, for more than 2 decades, generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using nonlymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas-mediated knockin mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and the morphology of lymphoid organs, as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control and mounted similar humoral immune responses as Y223F and wild-type animals. Collectively, our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase Cγ2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this posttranslational modification is dispensable for the function of BTK.

Undetectable off-target effects induced by FokI catalytic domain in mouse embryos.

The FokI catalytic domain can be fused to various DNA binding architectures to improve the precision of genome editing tools. However, evaluation of off-target effects is essential for developing these tools. We use Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) to detect low-frequency off-target editing events in mouse embryos injected with FokI-based architectures. Specifically, we test FokI-heterodimers fused with TALENs, FokI homodimers fused with RYdCas9, or FokI catalytic domains alone resulting in no significant off-target effects. These FokI genome editing systems exhibit undetectable off-target effects in mouse embryos, supporting the further development of these systems for clinical applications.

Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice.

Base editors have been reported to induce off-target mutations in cultured cells, mouse embryos and rice, but their long-term effects in vivo remain unknown. Here, we develop a Systematic evaluation Approach For gene Editing tools by Transgenic mIce (SAFETI), and evaluate the off-target effects of BE3, high fidelity version of CBE (YE1-BE3-FNLS) and ABE (ABE7.10F148A) in ~400 transgenic mice over 15 months. Whole-genome sequence analysis reveals BE3 expression generated de novo mutations in the offspring of transgenic mice. RNA-seq analysis reveals both BE3 and YE1-BE3-FNLS induce transcriptome-wide SNVs, and the numbers of RNA SNVs are positively correlated with CBE expression levels across various tissues. By contrast, ABE7.10F148A shows no detectable off-target DNA or RNA SNVs. Notably, we observe abnormal phenotypes including obesity and developmental delay in mice with permanent genomic BE3 overexpression during long-time monitoring, elucidating a potentially overlooked aspect of side effects of BE3 in vivo.

[Comparison of two-year stability between immediate implant action and delayed implantation for anterior teeth with periodontal disease].

PURPOSE: To investigate the feasibility of immediate implantation after extraction of anterior teeth in patients with periodontal disease and its clinical effect within 2 years. METHODS: Thirty patients (36 implants) who underwent anterior dental implant treatment for periodontal disease from 2016 to 2018 were randomly divided into immediate implantation group (17 implants) and delayed implantation group (19 implants). The patients were followed up for 2 years, the clinical parameters such as periodontal probing depth, pink esthetic score（PES）and implant neck bone resorption volume of implant neck were obtained. The data was statistically analyzed with SPSS 21.0 software package. RESULTS: During the 2-year follow-up period, no implant loss, and there was no significant difference in the depth of peri-implant probing between the two groups at each time point(P＞0.05). There was no significant difference in the volume of bone resorption at implant neck between the two groups(P>0.05). At 6, 12 and 24 months after completion of superstructure repair, there was no significant difference in pink esthetic score（PES）between the two groups (P＞0.05), but there was significant difference in pink esthetic score（PES) at the third month after restoration (P＜0.05). The immediate implantation group obtained more satisfactory soft tissue morphology around the implants. CONCLUSIONS: Under appropriate treatment conditions, there is no significant difference in implant success rate between immediate implantation and delayed implantation of anterior teeth in patients with periodontal disease. At the same time, it reduces the number of operations and shortens the course of treatment. In terms of soft tissue aesthetics, immediate implantation is slightly better than delayed implantation in the early stage after restoration, and can maintain a good soft tissue aesthetic effect.

Novel PI3K/Akt/mTOR signaling inhibitor, W922, prevents colorectal cancer growth via the regulation of autophagy.

W922, a novel PI3K/Akt/mTOR pathway inhibitor, exhibits efficient anti­tumor effects on HCT116, MCF­7 and A549 human cancer cells compared with other synthesized compounds. The present study aimed to investigate its anti­tumor effects on colorectal cancer cells. A total, of seven different colorectal cell lines were selected to test the anti­proliferation profile of W922, and HCT116 was found to be the most sensitive cell line to the drug treatment. W922 inhibited HCT116 cell viability and cell proliferation in vitro in concentration­ and time­dependent manners. Furthermore, W922 suppressed the tumor growth in a xenograft mouse model and exhibited low toxicity. The proteomic alterations in W922­treated HCT116 cells were found to be associated with cell cycle arrest, negative regulation of signal transduction and lysosome­related processes. W922 caused cell cycle arrest of HCT116 cells in G0­G1 phase, but only triggered slight apoptosis. In addition, the PI3K/Akt/mTOR signaling proteins were dephosphorylated upon W922 treatment. It has been reported that inhibition of mTOR is relevant to autophagy, and the present results also indicated that W922 was involved in autophagy induction. An autophagy inhibitor, chloroquine, was used to co­treat HCT116 cells with W922, and it was identified that the cell cycle arrest was impaired. Moreover, co­treatment of W922 and chloroquine led to a significant population of apoptotic cells, thus providing a promising therapeutic strategy for colorectal cancer.

CD36 and LC3B initiated autophagy in B cells regulates the humoral immune response.

Scavenger receptors are pattern recognition receptors that recognize both foreign and self-ligands, and initiate different mechanisms of cellular activation, often as co-receptors. The function of scavenger receptor CD36 in the immune system has mostly been studied in macrophages but it is also highly expressed by innate type B cells where its function is less explored. Here we report that CD36 is involved in macro-autophagy/autophagy in B cells, and in its absence, the humoral immune response is impaired. We found that CD36-deficient B cells exhibit a significantly reduced plasma cell formation, proliferation, mitochondrial mobilization and oxidative phosphorylation. These changes were accompanied by impaired initiation of autophagy, and we found that CD36 regulated autophagy and colocalized with autophagosome membrane protein MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). When we investigated T-cell-dependent immune responses, we found that mice with CD36 deficiency, specifically in B cells, exhibited attenuated germinal center responses, class switching, and antibody production as well as autophagosome formation. These findings establish a critical role for CD36 in B cell responses and may also contribute to our understanding of CD36-mediated autophagy in other cells as well as in B cell lymphomas that have been shown to express the receptor.Abbreviations: AICDA/AID: activation-induced cytidine deaminase; ATG5: autophagy related 5; ATP: adenosine triphosphate; BCR: B-cell receptor; CPG: unmethylated cytosine-guanosine; CQ: chloroquine; DC: dendritic cells; FOB: follicular B cells; GC: germinal center; Ig: immunoglobulin; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MZB: marginal zone B cells; NP-CGG: 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin; OCR: oxygen consumption rate; oxLDL: oxidized low-density lipoprotein; PC: plasma cells; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; SRBC: sheep red blood cells; Tfh: follicular helper T cells; TLR: toll-like receptor.

SOCS3 Expression by Thymic Stromal Cells Is Required for Normal T Cell Development.

The suppressor of cytokine signaling 3 (SOCS3) is a major regulator of immune responses and inflammation as it negatively regulates cytokine signaling. Here, the role of SOCS3 in thymic T cell formation was studied in Socs3fl/flActin-creER mice (Δsocs3) with a tamoxifen inducible and ubiquitous Socs3 deficiency. Δsocs3 thymi showed a 90% loss of cellularity and altered cortico-medullary organization. Thymocyte differentiation and proliferation was impaired at the early double negative (CD4-CD8-) cell stage and apoptosis was increased during the double positive (CD4+CD8+) cell stage, resulting in the reduction of recent thymic emigrants in peripheral organs. Using bone marrow chimeras, transplanting thymic organoids and using mice deficient of SOCS3 in thymocytes we found that expression in thymic stromal cells rather than in thymocytes was critical for T cell development. We found that SOCS3 in thymic epithelial cells (TECs) binds to the E3 ubiquitin ligase TRIM 21 and that Trim21-/- mice showed increased thymic cellularity. Δsocs3 TECs showed alterations in the expression of genes involved in positive and negative selection and lympho-stromal interactions. SOCS3-dependent signal inhibition of the common gp130 subunit of the IL-6 receptor family was redundant for T cell formation. Together, SOCS3 expression in thymic stroma cells is critical for T cell development and for maintenance of thymus architecture.

Targeting MARCO and IL37R on Immunosuppressive Macrophages in Lung Cancer Blocks Regulatory T Cells and Supports Cytotoxic Lymphocyte Function.

The progression and metastatic capacity of solid tumors are strongly influenced by immune cells in the tumor microenvironment. In non-small cell lung cancer (NSCLC), accumulation of anti-inflammatory tumor-associated macrophages (TAM) is associated with worse clinical outcome and resistance to therapy. Here we investigated the immune landscape of NSCLC in the presence of protumoral TAMs expressing the macrophage receptor with collagenous structure (MARCO). MARCO-expressing TAM numbers correlated with increased occurrence of regulatory T cells and effector T cells and decreased natural killer (NK) cells in these tumors. Furthermore, transcriptomic data from the tumors uncovered a correlation between MARCO expression and the anti-inflammatory cytokine IL37. In vitro studies subsequently showed that lung cancer cells polarized macrophages to express MARCO and gain an immune-suppressive phenotype through the release of IL37. MARCO-expressing TAMs blocked cytotoxic T-cell and NK-cell activation, inhibiting their proliferation, cytokine production, and tumor killing capacity. Mechanistically, MARCO+ macrophages enhanced regulatory T (Treg) cell proliferation and IL10 production and diminished CD8 T-cell activities. Targeting MARCO or IL37 receptor (IL37R) by antibody or CRISPR knockout of IL37 in lung cancer cell lines repolarized TAMs, resulting in recovered cytolytic activity and antitumoral capacity of NK cells and T cells and downmodulated Treg cell activities. In summary, our data demonstrate a novel immune therapeutic approach targeting human TAMs immune suppression of NK- and T-cell antitumor activities. SIGNIFICANCE: This study defines tumor-derived IL37 and the macrophage scavenger receptor MARCO as potential therapeutic targets to remodel the immune-suppressive microenvironment in patients with lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/4/956/F1.large.jpg.

Novel mouse model resistant to irreversible BTK inhibitors: a tool identifying new therapeutic targets and side effects.

Pharmacological inhibitors of Bruton tyrosine kinase (BTK) have revolutionized treatment of B-lymphocyte malignancies and show great promise for dampening autoimmunity. The predominant BTK inhibitors tether irreversibly by covalently binding to cysteine 481 in the BTK catalytic domain. Substitution of cysteine 481 for serine (C481S) is the most common mechanism for acquired drug resistance. We generated a novel C481S knock-in mouse model and, using a battery of tests, no overt B-lymphocyte phenotype was found. B lymphocytes from C481S animals were resistant to irreversible, but sensitive to reversible, BTK inhibitors. In contrast, irreversible inhibitors equally impaired T-lymphocyte activation in mice, mimicking the effect of treatment in patients. This demonstrates that T-lymphocyte blockage is independent of BTK. We suggest that the C481S knock-in mouse can serve as a useful tool for the study of BTK-independent effects of irreversible inhibitors, allowing for the identification of novel therapeutic targets and pinpointing potential side effects.

Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is an important pathogen of cattle. An attenuated live vaccine has recently been developed by this laboratory. However, an effective assay for the differentiation of infected from vaccinated animals (DIVA) is still lacking. Therefore, a comparative immunoproteomics study of the membrane and membrane associated proteins (MAPs) of M. bovis HB0801 and its attenuated strain (M. bovis-150) was aimed to identify potential antigens for DIVA assay. Triton-X-114 fractionated liposoluble proteins of both the virulent and attenuated strains were separated with 2-DE and proteins reacting with sera against the virulent M. bovis strain were detected by MS. A total of 19 differently expressed proteins were identified by MS, among them twelve proteins were detected by MALDI-TOF MS and seven antigenic proteins were identified by short-gun LC-MS/MS. Furthermore, these findings were confirmed at mRNA level by qRT-PCR. The results demonstrated that a putative lipoprotein encoded by functionally unknown gene Mbov_0730 (MbovP730) is a sensitive and specific antigen for DIVA assay. MbovP730 is absent in M. bovis-150 confirmed with Western blot assay and also didn't cross-react with other antisera against common pathogens including infectious bovine rhinotracheitis virus and bovine viral diarrhea virus by iELISA. Thereby rMbovP730-based iELISA was established. For clinical samples, this ELISA provided a sensitivity of 95.7% (95% CI: 90.4%, 98.2%) and specificity was 97.8% (95% CI: 88.4%, 99.6%). Antisera from vaccinated calves (n = 44) were found negative with rMbovP730 based iELISA, while positive with assays based on whole cell proteins of M. bovis-150 and M. bovis HB0801, respectively. In conclusion, this study identified the differential antigen MbovP730 between virulent and attenuated strains and established rMbovP730-based iELISA as a new DIVA method.

Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin.

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX-expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX- for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection.

A lack of knowledge regarding the antigenic properties of Mycoplasma bovis proteins prevents the effective control of bovine infections using immunological approaches. In this study, we detected and characterized a specific and sensitive M. bovis diagnostic biomarker. After M. bovis total proteins and membrane fractions were separated with two dimensional gel electrophoresis, proteins reacting with antiserawere detected using MALDI-TOF MS. Thirty-nine proteins were identified, 32 of which were previously unreported. Among them, immunoinformatics predicted eight antigens, encoded by Mbov_0106, 0116, 0126, 0212, 0275, 0579, 0739, and 0789, to have high immunological value. These genes were expressed in E. coli after mutagenesis of UGA to UGG using overlap extension PCR. A lipoprotein, MbovP579, encoded by a functionally unknown gene, was a sensitive and specific antigen for detection of antibodies in sera from both M. bovis-infected and vaccinated cattle. The specificity of MbovP579 was confirmed by its lack of cross-reactivity with other mycoplasmas, including Mycoplasma agalactiae. An iELISA based on rMbovP579 detected seroconversion 7 days post-infection (dpi). The ELISA had sensitivity of 90.2% (95% CI: 83.7%, 94.3%) and a specificity of 97.8% (95% CI: 88.7%, 99.6%) with clinical samples. Additional comparative studies showed that both diagnostic and analytic sensitivities of the ELISA were higher than those of a commercially available kit (p<0.01). We have thus detected and characterized the novel antigen, MbovP579, and established an rMbovP579-based ELISA as a highly sensitive and specific method for the early diagnosis of M. bovis infection.